Recombinant DNA technology has enabled expression of foreign (heterologous) polypeptides in microbial and other host cells. In yeast expression of heterologous polypeptides after transformation of yeast cells with suitable expression vectors comprising DNA sequences coding for said polypeptides has been successful for many species of polypeptides, such as insulin and insulin precursors, glucagon, glucagon like peptides and analogues thereof.
A common problem with expression of proteins or polypeptides of a limited size in a recombinant host is, however, proteolytic degradation of the expressed product by proteolytic enzymes produced by the host organism.
Thus, the isolated product may be a heterogeneous mixture of species of the desired polypeptide having different amino acid chain lengths. Another problem encountered in production of heterologous polypeptides in yeast may be low yield, presumably due to proteolytic processing both in intracellular compartments and at the plasma membrane caused by aberrant processing at internal sites in the polypeptide. Yeast contains a number of proteases used for processing yeast proteins e.g. Kex2p and Yps1p which cleave at the C-terminal side of a dibasic amino acid sequence, and the carboxypeptidase Kex1p which digests remaining basic amino acids after the endoproteolytic digestion by Kex2p, and Ste13p or Dap2p which cleave at X-Ala or X-Pro.
Some polypeptides, e.g. polypeptides having from about 10 to about 100 amino acids chains and none or a few disulphide bonds and/or are rich in basic amino acids, such as β-endorphine, glucagon and glucagon like peptides may be especially susceptible to intracellular and extracellular proteolytic degradation when expressed in a transformed host cell due to their short-chain open and non-disulfide stabilized structure resulting in an inhomogeneous product which may be proteolytically degraded in the N—and C-terminal ends as well as endoproteolytically degraded.
Furthermore, N-terminal cleavage of expressed polypeptides by host cell produced enzymes may cause decreased yield of a desired product with correct N-terminal if the N-terminal of the expressed product constitutes a cleavage site for endogenous enzymes. In yeast for example the enzyme Ste13p cleaves at X-Ala or X-Pro, where X can be any amino acid residue. Thus, polypeptides with an Ala or Pro residue as the second residue from the N-terminal end may be cleaved at the N-terminal end and the recovered polypeptide may be a mixture of different degradation products complicating the recovery process and reducing the overall yield.
Furthermore, small polypeptides with little tertiary structure and low content of α-helixes may have a higher tendency to form β-sheets that stack on each other and form fibrils during fermentation and down stream separation and purification steps in large scale production. Formation of fibrils may cause unwanted precipitation with loss of the desired product. Fibrillation may be prevented by treatment at high pH. However, such alkaline treatment is pretty harsh to the product and may cause unwanted formation of D-amino acids residues.
Human GLP-1 is a 37 amino acid residue peptide originating from preproglucagon which is synthesised in the L-cells in the distal ileum, in the pancreas and in the brain. Processing of preproglucagon to give GLP-1(7-36)amide, GLP-1(7-37) and GLP-2 occurs mainly in the L-cells. Both GLP-1 and GLP-2 have an Ala as the second amino acid residue from the N-terminal end and are thus prone for N-terminal cleavage when expressed in a host organism such as yeast.
Introduction of lipophilic acyl groups in naturally occurring peptides or analogues thereof has shown to lead to acylated peptides which have a protracted profile relative to the native peptide or unmodified analogues. The present invention provides a method for ensuring preferential acylation at the desired position in the polypeptide in question as it will appear from the following.